The escalating frequency of antibiotic resistant infections combined with the emergence of new infectious diseases underscores the critical need to develop novel antibiotics and better understand mechanisms of antibiotic resistance. This proposal addresses both of these needs through the described studies on enduracidin, a lipopeptide antibiotic produced by Streptomyces fungicidicus and an analog of the drug candidate ramoplanin. These peptides share a similar mechanism of action with vancomycin but bind the Lipid II target at a distinct site and are active against life-threatening vancomycin-resistant bacterial infections. The overall objectives of this proposal are to complete the functional characterization of the cloned enduracidin biosynthetic gene cluster and use this information to generate novel enduracidin analogs and elucidate the mechanism of resistance to the peptide in the producing organism. Functional analysis of the enduracidin biosynthetic gene cluster and flanking regions (aim 1) will employ a novel and rapid method of constructing a S. fungicidicus mutant library in which selected gene in the cluster and flanking regions is individually disrupted. This will permit a comprehensive analysis of individual gene loss-of-function on all aspects of antibiotic biosynthesis. The knowledge and tools acquired in aim 1 will drive the creation of enduracidin analogs using a combination of chemical and molecular genetics methods (aim 2). Novel peptides will be generated through gene disruption and mutational biosynthesis using exogenous and in situ generated precursors. Compounds will be prepared with variations in the lipid side chain that may have improved therapeutic properties or will serve as molecular tools for target analysis and kinetic studies (aim 2a). In aim 2b, gene disruption/complementation will be used to further examine alterations to the lipid tail, the role of peptide halogenation, and the function of various amino acid residues. Substitutions in the peptide core will be accomplished through gene swapping experiments designed to yield unique scaffolds for additional chemical modifications and also to explore the flexibility of the rare dual function epimerization/condensation domains in the enduracidin peptide synthetases. The goal of aim 3 is to identify the genetic elements in S. fungicidicus that confer self-resistance to the peptide and decipher the biochemical mechanism of immunity. In addition to producing novel antibiotics with potential as improved therapeutic agents for treating life- threatening Gram-positive bacterial infections, the knowledge and methods developed in this work will lead to new ways to increase natural product chemical diversity for drug discovery. Understanding the molecular basis of enduracidin resistance in the producing organism will help predict how new antibiotics may be affected by existing resistance mechanisms and provide insight into ways that resistance may emerge.